Compositions and methods for biological production of isoprene

Abstract

The present disclosure provides compositions and methods for biologically producing isoprene using methanotrophic bacteria that utilize carbon feedstock, such as methane or natural gas.

Description

STATEMENT REGARDING SEQUENCE LISTING[0001]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200206—411 WO_SEQUENCE_LISTING.txt. The text file is 58.5 KB, was created on Mar. 4, 2014, and is being submitted electronically via EFS-Web.BACKGROUND[0002]1. Technical Field[0003]The present disclosure provides compositions and methods for biologically producing isoprene, and more specifically, using methanotrophic bacteria to produce isoprene from carbon substrates, such as methane or natural gas.[0004]2. Description of the Related Art[0005]Isoprene, also known as 2-methyl-1,3-butadiene, is a volatile 5-carbon hydrocarbon. Isoprene is produced by a variety of organisms, including microbes, plants, and animal species (Kuzuyama, 2002, Biosci Biotechnol. Biochem. 66:1619-1627). There are two pathways for isoprene b...

AI Technical Summary

Benefits of technology

[0018]In another aspect, the present disclosure provides methods for screening mutant methanotrophic bacteria comprising: a) exposing the methanotrophic bacteria to a mutagen to produce mutant methanotrophic bacteria; b) transforming the mutant methanotrophic bacteria with exogenous nucleic acids encoding geranylgeranyl diphosphate synthase (GGPPS), phytoene synthase (CRTB), and phytoene dehydrogenase (CRTI); and c) culturing the mutant methanotrophic bacteria from step b) under conditions sufficient for growth; wherein a mutant methanotrophic bacterium that exhibits an increase in red pigmentation as compared to a reference methanotrophic bacterium that has not been exposed to a mutagen and has been transformed with exogenous nucleic acids encoding GGPPS, CRTB, and CRTI indicates that the mutant methanotrophic bacterium with increased red pigmentation exhibits increased isoprene precursor synthesis as compared to the reference methanotrophic bacterium. In certain embodiments, the mutagen is a radiation, a chemical, a plasmid, or a transposon. In certain embodiments, the mutant methanotrophic bacteria with increased red pigmentation or a clonal cell thereof is transformed with an exogenous nucleic acid encoding IspS. In further embodiments, at least one of the nucleic acids encoding GGPPS, CRTB, and CRTI is removed from or inactivated in the mutant methanotrophic bacterium with increased red pigmentation.

[0019]In yet another aspect, the present disclosure provides methods for screening isoprene pathway genes in methanotrophic bacteria comprising: a) transforming the methanotrophic bacteria with: i) at least one exogenous nucleic acid encoding an isoprene pathway enzyme; ii) exogenous nucleic acids encoding geranylgeranyl diphosphate synthase (GGPPS), phytoene synthase (CRTB), and phytoene dehydrogenase (CRTI); and b) culturing the methanotrophic bacteria from step a) under conditions sufficient for growth; wherein the transformed methanotrophic bacterium that exhibits an increase in red pigmentation as compared to a reference methanotrophic bacterium that has been transformed with exogenous nucleic acids encoding GGPPS, CRTB, and CRTI and does not contain the at least one exogenous nucleic acid encoding an isoprene pathway enzyme indicates that the at least one exogenous nucleic acid encoding an isoprene pathway enzyme confers increased isoprene precursor synthesis as compared to the reference methanotrophic bacterium. The isoprene pathway enzyme includes a DXP pathway enzyme or a mevalonate pathway enzyme. The at least one exogenous nucleic acid encoding an isoprene pathway enzyme may comprise a heterologous or homologous nucleic acid. The at least one exogenous nucleic acid encoding an isoprene pathway enzyme may be codon optimized for expression in the host methanotrophic bacteria. The homologous nucleic acid may be overexpressed in the methanotrophic bacteria. The at least one exogenous nucleic acid encoding an isoprene pathway enzyme may comprise a non-naturally occurring variant.

Problems solved by technology

Currently, isoprene's industrial use is limited by its tight supply.

Most synthetic rubbers are based on butadiene polymers, which is substantially more toxic than isoprene.

The isoprene yields from naturally producing organisms are not commercially attractive.

However, even with the use of relatively inexpensive cellulosic biomass as feedstock, more than half the mass of carbohydrate feedstocks is comprised of oxygen, which represents a significant limitation in conversion efficiency.

Isoprene and its derivatives (such as isoprenoids) have significantly lower oxygen content than the feedstocks, which limits the theoretical yield as oxygen must be eliminated as waste.

Thus, the economics of production of isoprene and its derivatives from carbohydrate feedstocks is prohibitively expensive.

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